Extranuclear Compartments Research Articles

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 440.

Zinc-Dependent Histone Deacetylases in Lung Endothelial Pathobiology.

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and, as such, provides a semi-selective barrier between the blood and the interstitial space. Compromise of the lung EC barrier due to inflammatory or toxic events may result in pulmonary edema, which is a cardinal feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). The EC functions are controlled, at least in part, via epigenetic mechanisms mediated by histone deacetylases (HDACs). Zinc-dependent HDACs represent the largest group of HDACs and are activated by Zn2+. Members of this HDAC group are involved in epigenetic regulation primarily by modifying the structure of chromatin upon removal of acetyl groups from histones. In addition, they can deacetylate many non-histone histone proteins, including those located in extranuclear compartments. Recently, the therapeutic potential of inhibiting zinc-dependent HDACs for EC barrier preservation has gained momentum. However, the role of specific HDAC subtypes in EC barrier regulation remains largely unknown. This review aims to provide an update on the role of zinc-dependent HDACs in endothelial dysfunction and its related diseases. We will broadly focus on biological contributions, signaling pathways and transcriptional roles of HDACs in endothelial pathobiology associated mainly with lung diseases, and we will discuss the potential of their inhibitors for lung injury prevention.

Characterizing the differential distribution and targets of Sumo1 and Sumo2 in the mouse brain

SUMOylation is an evolutionarily conserved eukaryotic posttranslational protein modification with broad biological relevance. Differentiating between the major small ubiquitin-like modifier (SUMO) paralogs and uncovering paralog-specific functions invivo has long been very difficult. To overcome this problem, we generated His6-HA-Sumo2 and HA-Sumo2 knockin mouse lines, expanding upon our existing His6-HA-Sumo1 mouse line, to establish a "toolbox" for Sumo1-Sumo2 comparisons invivo. Leveraging the specificity of the HA epitope, we performed whole-brain imaging and uncovered regional differences between Sumo1 and Sumo2 expression. At the subcellular level, Sumo2 was specifically detected in extranuclear compartments, including synapses. Immunoprecipitation coupled with mass spectrometry identified shared and specific neuronal targets of Sumo1 and Sumo2. Target validation using proximity ligation assays provided further insight into the subcellular distribution of neuronal Sumo2-conjugates. The mouse models and associated datasets provide a powerful framework to determine the native SUMO "code" in cells of the central nervous system.

Role of nucleic acid sensing in the pathogenesis of type 1 diabetes.

During infections, nucleic acids of pathogens are also engaged in recognition via several exogenous and cytosolic pattern recognition receptors, such as the toll-like receptors, retinoic acid inducible gene-I-like receptors, and nucleotide-binding and oligomerization domain-like receptors. The binding of the pathogen-derived nucleic acids to their corresponding sensors initiates certain downstream signaling cascades culminating in the release of type-I interferons (IFNs), especially IFN-α and other cytokines to induce proinflammatory responses towards invading pathogens leading to their clearance from the host. Although these sensors are hardwired to recognize pathogen associated molecular patterns, like viral and bacterial nucleic acids, under unusual physiological conditions, such as excessive cellular stress and increased apoptosis, endogenous self-nucleic acids like DNA, RNA, and mitochondrial DNA are also released. The presence of these self-nucleic acids in extranuclear compartments or extracellular spaces or their association with certain proteins sometimes leads to the failure of discriminating mechanisms of nucleic acid sensors leading to proinflammatory responses as seen in autoimmune disorders, like systemic lupus erythematosus, psoriasis and to some extent in type 1 diabetes (T1D). This review discusses the involvement of various nucleic acid sensors in autoimmunity and discusses how aberrant recognition of self-nucleic acids by their sensors activates the innate immune responses during the pathogenesis of T1D.

Activation of ATM kinase by ROS generated during ionophore-induced mitophagy in human T and B cell malignancies.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor, also possesses non-nuclear functions owing to its presence in extra-nuclear compartments, including peroxisomes, lysosomes, and mitochondria. ATM is frequently altered in several human cancers. Recently, we and others have shown that loss of ATM is associated with defective mitochondrial autophagy (mitophagy) in ataxia-telangiectasia (A-T) fibroblasts and B-cell lymphomas. Further, we reported that ATM protein but not ATM kinase activity is required for mitophagy. However, the mechanism of ATM kinase activation during ionophore-induced mitophagy is unknown. In the work reported here, using several ionophores in A-T and multiple T-cell and B-cell lymphoma cell lines, we show that ionophore-induced mitophagy triggers oxidative stress-induced ATMSer1981 phosphorylation through ROS activation, which is different from neocarzinostatin-induced activation of ATMSer1981, Smc1Ser966, and Kap1Ser824. We used A-T cells overexpressed with WT or S1981A (auto-phosphorylation dead) ATM plasmids and show that ATM is activated by ROS-induced oxidative stress emanating from ionophore-induced mitochondrial damage and mitophagy. The antioxidants N-acetylcysteine and glutathione significantly inhibited ROS production and ATMSer1981 phosphorylation but failed to inhibit mitophagy as determined by retroviral infection with mt-mKeima construct followed by lysosomal dual-excitation ratiometric pH measurements. Our data suggest that while ATM kinase does not participate in mitophagy, it is activated via elevated ROS.

Sub-cellular localization specific SUMOylation in the heart

Although the majority of SUMO substrates are localized in the nucleus, SUMOylation is not limited to nuclear proteins and can be also detected in extra-nuclear proteins. In this review, we will highlight and discuss how SUMOylation in different cellular compartments regulate biological processes. First, we will discuss the key role of SUMOylation of proteins in the extra-nuclear compartment in cardiomyocytes, which is overwhelmingly cardio-protective. On the other hand, SUMOylation of nuclear proteins is generally detrimental to the cardiac function mainly because of the trans-repressive nature of SUMOylation on many transcription factors. We will also discuss the potential role of SUMOylation in epigenetic regulation. In this review, we will propose a new concept that shuttling of SUMO proteases between the nuclear and extra-nuclear compartments without changing their enzymatic activity regulates the extent of SUMOylation in these compartments and determines the response and fate of cardiomyocytes after cardiac insults. Approaches focused specifically to inhibit this shuttling in cardiomyocytes will be necessary to understand the whole picture of SUMOylation and its pathophysiological consequences in the heart, especially after cardiac insults. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

Psychosis Risk Candidate ZNF804A Localizes to Synapses and Regulates Neurite Formation and Dendritic Spine Structure

BackgroundVariation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia and bipolar disorder. Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed. MethodsThe localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells, human induced pluripotent stem cells, or in primary rat cortical neurons. In addition, small interfering RNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology, and responses to activity-dependent stimulations. ResultsEndogenous ZNF804A protein localized to somatodendritic compartments and colocalized with the putative synaptic markers in young neurons derived from human neural progenitor cells and human induced pluripotent stem cells. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and colocalized with synaptic proteins in specific nanodomains, as determined by super-resolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density and impaired responses to activity-dependent stimulation. ConclusionsThese data reveal a novel subcellular distribution for ZNF804A within somatodendritic compartments and a nanoscopic organization at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines, and activity-dependent structural plasticity.

P150 L’effet antiprolifératif de l’isomère trans9, trans11 de l’acide linoléique conjugué sur les cellules du cancer du sein MCF-7 est associé à une activation du facteur nucléaire LXR

The novel ARF-like GTPase ARL7 is a close relative of ARL4 and ARL6 (71% and 59% identical amino acids). A striking characteristic of these GTPases is their basic C-terminus which, when fused to the C-terminus of green fluorescent protein (GFP), targets the constructs to the nucleus of transfected COS-7 cells. Full length ARL4 was detected in both nuclear and extranuclear compartments, whereas a construct of ARL4 lacking its C-terminus was excluded from the nucleus. Nucleotide exchange rates of recombinant ARL4, ARL6 and ARL7 were similar and appeared considerably higher than those of other members of the ARF family (ARF1, ARP). It is concluded that ARL4, ARL6 and ARL7 form a subgroup within the ARF family with similar, possibly nuclear, function.

Mass spectrometry-based functional proteomics of poly(ADP-ribose) polymerase-1

PARP-1 is an abundant nuclear protein that plays an essential role in the regulation of many genome integrity and chromatin-based processes, such as DNA repair, replication or transcriptional regulation. PARP-1 modulates the function of chromatin and nuclear proteins through several poly(ADP-ribose) (pADPr)-dependent pathways. Aside from the clearly established role of PARP-1 in the maintenance of genome stability, PARP-1 also emerged as an important regulator that links chromatin functions with extranuclear compartments. pADPr signaling has notably been found to be responsible for PARP-1-mediated mitochondrial dysfunction and cell death. Defining the mechanisms that govern the intrinsic functions of PARP-1 is fundamental to the understanding of signaling networks regulated by pADPr. The emergence of mass spectrometry-based proteomics and its broad applications in the study of biological systems represents an outstanding opportunity to widen our knowledge of the functional spectrum of PARP-1. In this article, we summarize various PARP-1 targeted proteomics studies and proteome-wide analyses that shed light on its protein interaction partners, expression levels and post-translational modifications.

Current and Future Development of Estrogen Receptor Ligands: Applications in Estrogen-Related Cancers

17β;-Estradiol (E2), via its cognate receptors (ERs), regulates several aspects of human physiology including development, reproduction and tissue homeostasis. Consequently, E2 could also be implicated in the development or progression of several pathologies, including cancer. Two different ER subtypes are present in mammals (ERβ and ERβ), which display specific roles in E2-related cancers, different tissue distribution, and multiple action mechanisms (i.e., genomic and extranuclear mechanisms). Here, the complex pattern of the relative contribution of each ER subtype in the E2- dependent cancers has been summarized by taking into consideration the molecular events which occur both in the nucleus and in extranuclear compartments. In the second part of this paper, we reviewed the current literature available on the drug-targeting of the ERs, as well as the recent literature and patents describing new and upcoming molecules. These new molecules will probably greatly improve the repertoire of anti-hormonal therapeutic strategies. However, further new drug design programs, which should include all molecular mechanisms at the basis of ER biology, are needed to expand the anti-ER treatments in new and more efficient therapeutic directions. This review also outlines relevant patents.

Progesterone and estrogen receptor expression and activity in human non-small cell lung cancer

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. Although it is clear that tobacco smoking is a major cause of lung cancer, about half of all women with lung cancer worldwide are never-smokers. Despite a declining smoking population, the incidence of non-small cell lung cancer (NSCLC), the predominant form of lung cancer, has reached epidemic proportions particularly in women. Emerging data suggest that factors other than tobacco, namely endogenous and exogenous female sex hormones, have a role in stimulating NSCLC progression. Aromatase, a key enzyme for estrogen biosynthesis, is expressed in NSCLC. Clinical data show that women with high levels of tumor aromatase (and high intratumoral estrogen) have worse survival than those with low aromatase. The present and previous studies also reveal significant expression and activity of estrogen receptors (ERα, ERβ) in both extranuclear and nuclear sites in most NSCLC. We now report further on the expression of progesterone receptor (PR) transcripts and protein in NSCLC. PR transcripts were significantly lower in cancerous as compared to non-malignant tissue. Using immunohistochemistry, expression of PR was observed in the nucleus and/or extranuclear compartments in the majority of human tumor specimens examined. Combinations of estrogen and progestins administered in vitro cooperate in promoting tumor secretion of vascular endothelial growth factor and, consequently, support tumor-associated angiogenesis. Further, dual treatment with estradiol and progestin increased the numbers of putative tumor stem/progenitor cells. Thus, ER- and/or PR-targeted therapies may offer new approaches to manage NSCLC.

A Neurotoxic Phosphoform of Elk-1 Associates with Inclusions from Multiple Neurodegenerative Diseases

Neurodegenerative diseases are characterized by a number of features including the formation of inclusions, early synaptic degeneration and the selective loss of neurons. Molecules serving as links between these shared features have yet to be identified. Identifying candidates within the diseased microenvironment will open up novel avenues for therapeutic intervention. The transcription factor Elk-1 resides within multiple brain areas both in nuclear and extranuclear neuronal compartments. Interestingly, its de novo expression within a single dendrite initiates neuronal death. Given this novel regionalized function, we assessed whether extranuclear Elk-1 and/or phospho-Elk-1 (pElk-1) protein might be associated with a spectrum of human neurodegenerative disease cases including Lewy body Disease (e.g. Parkinson's), Alzheimer's disease, and Huntington's Disease. We first determined the importance of Elk-1 post-translational modifications on its ability to initiate regionalized cell death. We next screened human cases from three major neurodegenerative diseases to look for remarkable levels of Elk-1 and/or pElk-1 protein as well as their association with inclusions characteristic of these diseases. We compared our findings to age-matched control cases. We find that the ability of Elk-1 to initiate regionalized neuronal death depends on a specific phosphosite, T417. Furthermore, we find that T417+ Elk-1 uniquely associates with several types of inclusions present in cases of human Lewy body Disease, Alzheimer's disease, and Huntington's Disease. These results suggest a molecular link between the presence of inclusions and neuronal loss that is shared across a spectrum of neurodegenerative disease.

Structural and functional analysis of fatty acid-binding proteins

The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis.

The Emerging Functions and Mechanisms of Mammalian Fatty Acid–Binding Proteins

Fatty acid-binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids with high affinity. Nine separate mammalian FABPs have been identified, and their tertiary structures are highly conserved. The FABPs have unique tissue-specific distributions that have long suggested functional differences among them. In the last decade, considerable progress has been made in understanding the specific functions of the FABPs and, in some cases, their mechanisms of action at the molecular level. The FABPs appear to be involved in the extranuclear compartments of the cell by trafficking their ligands within the cytosol via interactions with organelle membranes and specific proteins. Several members of the FABP family have been shown to function directly in the regulation of cognate nuclear transcription factor activity via ligand-dependent translocation to the nucleus. This review will focus on these emerging functions and mechanisms of the FABPs, highlighting the unique functional properties of each as well as the similarities among them.

Proteomimetic Cell Penetrating Peptides

QSAR analysis of the primary sequence of human cytochrome c indicated that numerous putative cell penetrating peptide sequences are located within major amino- and carboxyl-terminal helical domains. Two such sequences, Cyt c77−101 and the shorter homologue Cyt c86−101, readily translocate the plasma membrane of U373MG astrocytoma cells but differentially accumulate in extranuclear or nuclear compartments. Such sequences could be employed for the selective intracellular targeting of cytotoxins and other bioactive cargoes. Moreover, these peptides also induce apoptosis, indicating that they mimic the role of cytochrome c as a key regulator of programmed cell death. We propose that these domains may constitute an integral transduction domain within cytochrome c that enables the protein to directly translocate plasma membranes. These, and other studies, indicate that cell penetrating peptides (CPP) are valuable tools both for the purposes of intracellular delivery and for the direct manipulation of therapeutically relevant proteins and cellular processes. We propose that the term bioportide should be introduced to distinguish biologically active, proteomimetic CPP from inert sequences that include the majority of common vectors.

Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone β receptor

Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B signaling pathway has been associated with multiple human cancers. Recently we showed that AKT is activated in both the thyroid and metastatic lesions of a mouse model of follicular thyroid carcinoma [thyroid hormone beta receptor (TRbeta)(PV/PV) mice]. This TRbeta(PV/PV) mouse harbors a knock-in mutant TRbeta gene (TRbetaPV mutant) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. Here we show that in thyroid tumors, PV mutant bound significantly more to the PI3K-regulatory subunit p85alpha, resulting in a greater increase in the kinase activity than did TRbeta1 in wild-type mice. By GST pull-down assays, the ligand-binding domain of TR was identified as the interaction site with p85alpha. By confocal fluorescence microscopy, p85alpha was shown to colocalize with TRbeta1 or PV mainly in the nuclear compartment of cultured tumor cells from TRbeta(PV/PV) mice, but cytoplasmic p85alpha/PV or p85alpha/TRbeta1 complexes were also detectable. Further biochemical analysis revealed that the activation of the PI3K-AKT-mammalian target of the rapamycin-p70(S6K) pathway was observed in both the cytoplasmic and nuclear compartments, whereas the activation of the PI3K-integrin-linked kinase-matrix metalloproteinase 2 pathway was detected mainly in the extranuclear compartments. These results suggest that PV, via the activation of p85alpha, could act to affect PI3K downstream signaling in both the nuclear and extranuclear compartments, thereby contributing to thyroid carcinogenesis. Importantly, the present study unveils a mechanism by which a mutant TR acts to activate PI3K activity via protein-protein interactions.

Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer

Regulation of breast cancer growth by estrogen is mediated by estrogen receptors (ER) in nuclear and extranuclear compartments. We assessed the structure and functions of extranuclear ER that initiate downstream signaling to the nucleus. ER, including full-length 66-kDa ER and a 46-kDa ER splice variant, are enriched in lipid rafts from MCF-7 cells with (MCF-7/HER-2) or without (MCF-7/PAR) HER-2 gene overexpression and co-localize with HER-1 and HER-2 growth factor receptors, as well as with lipid raft marker flotillin-2. In contrast, ER-negative MCF-7 cells do not express nuclear or lipid raft ER. ER knockdown with siRNA also elicits a marked loss of ER in MCF-7 cell rafts. In MCF-7/PAR cells, estrogen enhances ER association with membrane rafts and induces rapid phosphorylation of nuclear receptor coactivator AIB1, actions not detected in ER-negative cells. Thus, nuclear and lipid raft ER derive from the same transcript, and extranuclear ER co-localizes with HER receptors in membrane signaling domains that modulate downstream nuclear events leading to cell growth.

Use of mass spectrometry-based lipidomics to probe PITPα (phosphatidylinositol transfer protein α) function inside the nuclei of PITPα+/+ and PITPα−/− cells

The mammalian phospholipid exchange protein PITPalpha (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPalpha ablation in mice, PITPalpha(-/-) MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d(9) and inositol-d(6), into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPalpha to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d(6) incorporation into whole-cell PtdIns species was consistently higher in PITPalpha(-/-) MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d(6) was apparent in the PITPalpha(-/-) cells and mirrored that in PITPalpha(+/+) cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPalpha can be accommodated by other mechanisms.

Impairment of phosphatase 2A contributes to the prolonged MAP kinase phosphorylation in Alzheimer's disease fibroblasts

The serine/threonine phosphatase 2A (PP2A) has been implicated in the pathogenesis of Alzheimer's disease (AD) due to its important role in regulating dephosphorylation of the microtubule-associated protein tau and mitogen-activated protein (MAP) kinase. In the present study, we show that PP2A was responsible for dephosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) following its activation by BK stimulation. Abnormal gene and protein expressions of PP2A, as well as its activity, were found to contribute to the abnormally prolonged Erk1/2 phosphorylation in the AD fibroblasts. Inhibition of PP2A with okadiac acid produced enhanced and more lasting Erk1/2 phosphorylation after BK stimulation, whereas FK506, an inhibitor of PP2B and FK-binding protein, inhibited the BK-stimulated Erk1/2 phosphorylation. Furthermore, while the phosphorylated Erk1/2 was concentrated in the nucleus of AC cells, it was mainly distributed in the extranuclear compartments of AD cells. These results suggest that the delayed dephosphorylation of Erk1/2 in AD cells following its BK-stimulated activation may be due to deficits of PP2A activity and impaired nuclear translocation of phosphorylated Erk1/2.

Sex differences in androgen and estrogen receptor expression in rat substantia nigra during development: an immunohistochemical study

Gonadal hormones are important regulators of sexual differentiation of the CNS. Exposure to testosterone and estrogen during development causes permanent organizational differences between males and females. We previously described functional sex-related differences of the GABA Aergic circuits of the rat substantia nigra pars reticulata (SNR) involved in the control of flurothyl seizures. This sexual differentiation of the SNR is regulated by postnatal testosterone. To assess whether the organizing effects of testosterone in the SNR are mediated via the androgen receptor (AR) and/or estrogen receptors (ER), we used immunohistochemistry to study the ontogeny of AR, ERα and ERβ expression in SNR and substantia nigra pars compacta (SNC) of male and female rats. Rats on the day of birth [postnatal day (PN) 0] and at PN1, PN5, PN15 and PN30 were used. AR- and ERβ-immunopositive cells were present in SNR and SNC in both sexes and at all ages. ERα was not detected in male and female SNC at PN0–PN1. In both substantia nigra (SN) regions, there were developmentally regulated sex differences in AR, ERα and ERβ immunoreactivity. In the SN, each receptor showed specific intracellular localization: AR was present in the nucleus, ERα and ERβ were present both in nuclear and extranuclear compartments. ERα was detected also in processes. At PN0–PN1, quantitative analysis revealed sex and regional differences in the distribution of SN cells expressing AR and ERα, while ERβ were equally present in both sexes. The presence of gonadal steroid receptors in the SN suggests that the biological effects of gonadal hormones in the CNS extend beyond reproduction-related functions and may affect and modify motor behaviors (including seizures) in a sex-specific manner. Based on the ontogeny of SNR ERβ, we hypothesize that postnatal injections of testosterone may regulate the nigral GABA A system through the aromatization pathway and activation of ERβ.